Abstract
BackgroundRhabdomyosarcoma (RMS) is a highly malignant tumour accounting for nearly half of soft tissue sarcomas in children. MicroRNAs (miRNAs) represent a class of short, non-coding, regulatory RNAs which play a critical role in different cellular processes. Altered miRNA levels have been reported in human cancers, including RMS.MethodsUsing deep sequencing technology, a total of 685 miRNAs were investigated in a group of alveolar RMSs (ARMSs), embryonal RMSs (ERMSs) as well as in normal skeletal muscle (NSM). Q-PCR, MTT, cytofluorimetry, migration assay, western blot and immunofluorescence experiments were carried out to determine the role of miR-378a-3p in cancer cell growth, apoptosis, migration and differentiation. Bioinformatics pipelines were used for miRNA target prediction and clustering analysis.ResultsNinety-seven miRNAs were significantly deregulated in ARMS and ERMS when compared to NSM. MiR-378 family members were dramatically decreased in RMS tumour tissue and cell lines. Interestingly, members of the miR-378 family presented as a possible target the insulin-like growth factor receptor 1 (IGF1R), a key signalling molecule in RMS. MiR-378a-3p over-expression in an RMS-derived cell line suppressed IGF1R expression and affected phosphorylated-Akt protein levels. Ectopic expression of miR-378a-3p caused significant changes in apoptosis, cell migration, cytoskeleton organization as well as a modulation of the muscular markers MyoD1, MyoR, desmin and MyHC. In addition, DNA demethylation by 5-aza-2′-deoxycytidine (5-aza-dC) was able to up-regulate miR-378a-3p levels with a concomitant induction of apoptosis, decrease in cell viability and cell cycle arrest in G2-phase. Cells treated with 5-aza-dC clearly changed their morphology and expressed moderate levels of MyHC.ConclusionsMiR-378a-3p may function as a tumour suppressor in RMS and the restoration of its expression would be of therapeutic benefit in RMS. Furthermore, the role of epigenetic modifications in RMS deserves further investigations.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-880) contains supplementary material, which is available to authorized users.
Highlights
Rhabdomyosarcoma (RMS) is a highly malignant tumour accounting for nearly half of soft tissue sarcomas in children
RNA was prepared from four alveolar RMSs (ARMSs) and four embryonal RMSs (ERMSs), and from a pool of normal skeletal muscle (NSM) obtained from eight donors
Tumour miRNAs on average were more represented in the intermediate abundance class (102104 Counts Per Millions (CPM)), whilst they were under-represented in the lowest (1-102 CPM) and in the highest abundance (>104 CPM) classes
Summary
Rhabdomyosarcoma (RMS) is a highly malignant tumour accounting for nearly half of soft tissue sarcomas in children. Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood [1], representing approximately 50% of all sarcomas in children aged 0–14 years and 4-5% of malignant solid tumours in the paediatric population. The role of genetic factors in the development of RMS has been confirmed by several recent epidemiological observations and advances in molecular genetics. ARMS and ERMS are both characterised by particular genetic alterations that are likely to play a decisive role in cancer pathogenesis. Loss of heterozygosity of the short arm of chromosome 11 (11p15.5), with over-expression of the insulin-like growth factor II, is often associated with ERMS [10]. Several tumour causative genes have been identified, a detailed understanding of the molecular mechanisms underlying RMS development has not yet been achieved
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